Superdex 200 increase 3.2 300

Size-exclusion chromatography with Superdex-200 Increase 3.2/300 (GE Healthcare) was conducted in the buffer (25 mM Tris/HCl pH 7.8, 5 mM MgCl₂, 4 mM 2-mercaptoethanol, 5% ethylene glycol). The absorption of proteins was detected at 489 and 280 nm. The illuminated samples (R) were pre-illuminated with 655 nm LED light for 5 min before injection. For each run, 24 µl of sample mixture (5 mg/ml each) was injected and eluted at 70 µl/min. The molecular weight estimates were determined by calculating a standard curve of marker proteins Vitamin B12 (1.35 kDa) myoglobin (17 kDa), ovalbumin (44 kDa), γ-globulin (158 kDa), and thyroglobulin (670 kDa).

1.2 μM purified DDK was incubated with 1.4 μM purified Rad53 variant in 32 mM HEPES-KOH pH 7.6/200 mM KOAc/10 mM Mg(OAc)₂ / 0.5 mM EDTA/0.5 mM EGTA/0.01 % NP-40 substitute/10 % glycerol/1 mM AMP-PNP/1 mM DTT for 30 min at 30°C. The sample was fractionated on a Superdex 200 Increase 3.2/300 (GE Healthcare) gel filtration column in 25 mM HEPES-KOH pH 7.6/10 mM Mg(OAc)₂/0.02 % NP-40 substitute/5 % glycerol/185 mM KOAc/1 mM DTT/1 mM AMP-PNP. Elution fractions were analyzed by SDS-PAGE and Coomassie stain or western blotting.

TmrAB was reconstituted in lipid nanodiscs (Hofmann et al., 2019 (link)) composed of biotinylated MSP1D1. For biotinylation, purified MSP1D1 was buffer-exchanged to 20 mM HEPES-NaOH pH 7.5, 150 mM NaCl using Zeba spin desalting column (Thermo Fisher). A four-fold molar excess of EZ-Link NHS-PEG₄-biotin (Thermo Fisher) was added for 2 hr on ice. Residual NHS-PEG₄-biotin was quenched by adding 10 mM of Tris–HCl pH 8.0. To remove the excess of NHS-PEG₄-biotin, samples were buffer exchanged to 20 mM HEPES-NaOH pH 7.5, 150 mM NaCl using PD-10 desalting column (GE Healthcare). Bovine brain lipids (Sigma-Aldrich) were solubilized in 20 mM of β-DDM. Biotinylated MSP1D1 was mixed with Bovine brain lipids with and without TmrAB in a TmrAB/MSP1D1/lipid molar ratio of 1/7.5/100 in SEC buffer without detergent to form TmrAB-filled or empty liposomes, respectively. Samples were incubated for 30 min at 20°C, and SM-2 Bio-beads (Bio-Rad) were added in two steps at 4°C (1 hr and overnight) for detergent removal. Samples were concentrated using Amicon Ultra-0.5 ml centrifugal filters with 50 kDa cut-off (Merck Millipore). Reconstituted nanodiscs were isolated by size-exclusion chromatography (SEC) via Superdex 200 Increase 3.2/300 (GE Healthcare).

Construction of the pSA4 plasmids for recombinant expression of ABCE1, aRF1, aPelota, and aIF6 from S. solfataricus in E. coli was described previously (Barthelme et al, 2007, 2011). All proteins were expressed, purified, and stored as previously described (Nürenberg‐Goloub et al, 2018). Protein quality was assured by SDS–PAGE and size exclusion chromatography (Superdex 200 Increase 3.2/300, GE Healthcare) in SEC buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM β‐mercaptoethanol) at 4°C recording absorption at 280 and 410 nm to monitor FeSD cluster integrity.

Analytical SEC experiments were performed on a Superdex 200 Increase 3.2/300 or a Superose 6 Increase 3.2/300 column (GE Healthcare). To detect the formation of a complex, proteins were mixed at equimolar ratios and incubated for 1 hr on ice before SEC. All samples were eluted under isocratic conditions at 4°C in SEC buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 5% glycerol]. Elution of proteins was monitored by absorbance at 280 nm. 100 µl fractions were collected and analyzed by SDS-PAGE and Coomassie staining.